CD123-binding chimeric antigen receptors

Abstract

Disclosed are compositions and methods for targeted treatment of CD123-expressing cancers. In particular, chimeric antigen receptor (CAR) polypeptides are disclosed that can be used with adoptive cell transfer to target and kill CD123-expressing cancers. Also disclosed are immune effector cells, such as T cells or Natural Killer (NK) cells, that are engineered to express these CARs. Therefore, also disclosed are methods of providing an anti-tumor immunity in a subject with a CD123-expressing cancer that involves adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CARs.

What is claimed is: 
     
       1. A chimeric antigen receptor (CAR) polypeptide, comprising a CD123 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region, wherein the CD123 antigen binding domain is a single-chain variable fragment (scFv) of an antibody comprising a variable heavy (V H ) domain having CDR1, CDR2 and CDR3 sequences and a variable light (V L ) domain having CDR1, CDR2 and CDR3 sequences, and wherein
 (a) the CDR1 sequence of the V H  domain comprises the amino acid sequence GYTFTDYN (SEQ ID NO:1), the CDR2 sequence of the V H  domain comprises the amino acid sequence INPNNGGT (SEQ ID NO:2), the CDR3 sequence of the V H  domain comprises the amino acid sequence ARKGYGGNYDYFDY (SEQ ID NO:3), the CDR1 sequence of the V L  comprises the amino acid sequence QSIGTS (SEQ ID NO:4), the CDR2 sequence of the V L  domain comprises the amino acid sequence YASx (SEQ ID NO:5), and the CDR3 sequence of the V L  domain comprises the amino acid sequence QQSNSWPYT (SEQ ID NO:6); or 
 (b) the CDR1 sequence of the V H  domain comprises the amino acid sequence GFNIKDTY (SEQ ID NO:7), the CDR2 sequence of the V H  domain comprises the amino acid sequence IDPANGNT (SEQ ID NO:9), the CDR3 sequence of the V H  domain comprises the amino acid sequence ALYYYGGSLDY (SEQ ID NO:11), the CDR1 sequence of the V L  comprises the amino acid sequence QSLLYSGNQKNY (SEQ ID NO:13), the CDR2 sequence of the V L  domain comprises the amino acid sequence WASx (SEQ ID NO:14), and the CDR3 sequence of the V L  domain comprises the amino acid sequence QQYYSYPRT (SEQ ID NO:15); or 
 (c) the CDR1 sequence of the V H  domain comprises the amino acid sequence GFSLSTYGMG (SEQ ID NO:8), the CDR2 sequence of the V H  domain comprises the amino acid sequence IYWDDDK (SEQ ID NO:10), the CDR3 sequence of the V H  domain comprises the amino acid sequence AQSLIYDGYYGFAY (SEQ ID NO:12), the CDR1 sequence of the V L  comprises the amino acid sequence QSLLYSGNQKNY (SEQ ID NO:13), the CDR2 sequence of the V L  domain comprises the amino acid sequence WASx (SEQ ID NO:14), and the CDR3 sequence of the V L  domain comprises the amino acid sequence QQYYSYPRT (SEQ ID NO:15); or 
 (d) the CDR1 sequence of the V H  domain comprises the amino acid sequence GYTFTYYG (SEQ ID NO:16), the CDR2 sequence of the V H  domain comprises the amino acid sequence INTYSGVP (SEQ ID NO:17), the CDR3 sequence of the V H  domain comprises the amino acid sequence ARWIYYSDLYGMDY (SEQ ID NO:18), the CDR1 sequence of the V L  comprises the amino acid sequence QSIVHSNGDTY (SEQ ID NO:19), the CDR2 sequence of the V L  domain comprises the amino acid sequence KVSx (SEQ ID NO:20), and the CDR3 sequence of the V L  domain comprises the amino acid sequence FQGSHVPWT (SEQ ID NO:21); or 
 (e) the CDR1 sequence of the V H  domain comprises the amino acid sequence GYTFSSYW (SEQ ID NO:22), the CDR2 sequence of the V H  domain comprises the amino acid sequence INPSSGYT (SEQ ID NO:24), the CDR3 sequence of the V H  domain comprises the amino acid sequence ARDGNYDHWYFDV (SEQ ID NO:26), or 
 the CDR1 sequence of the V H  domain comprises the amino acid sequence GYTLTTYL (SEQ ID NO:23), the CDR2 sequence of the V H  domain comprises the amino acid sequence INPNSGSS (SEQ ID NO:25), the CDR3 sequence of the V H  domain comprises the amino acid sequence AIRHYGGSLFDY (SEQ ID NO:27), and 
 the CDR1 sequence of the V L  comprises the amino acid sequence QDINSY (SEQ ID NO:28), the CDR2 sequence of the V L  domain comprises the amino acid sequence RANx (SEQ ID NO:30), and the CDR3 sequence of the V L  domain comprises the amino acid sequence LQYDELLT (SEQ ID NO:31), or 
 the CDR1 sequence of the V L  comprises the amino acid sequence QSLLNSRTRKNY (SEQ ID NO:29), the CDR2 sequence of the V L  domain comprises the amino acid sequence RANx (SEQ ID NO:30), and the CDR3 sequence of the V L  domain comprises the amino acid sequence EQSYNLFT (SEQ ID NO:32). 
 
     
     
       2. The polypeptide of  claim 1 ,
 wherein the anti-CD123 scFv V H  domain comprises the amino acid sequence SEQ ID NO:33, and wherein the anti-CD123 scFv V L  domain comprises the amino acid sequence SEQ ID NO:39; 
 wherein the anti-CD123 scFv V H  domain comprises the amino acid sequence SEQ ID NO:34 or SEQ ID NO:35, and wherein the anti-CD123 scFv V L  domain comprises the amino acid sequence SEQ ID NO:40; 
 wherein the anti-CD123 scFv V H  domain comprises the amino acid sequence SEQ ID NO:36, and wherein the anti-CD123 scFv V L  domain comprises the amino acid sequence SEQ ID NO:41; 
 wherein the anti-CD123 scFv V H  domain comprises the amino acid sequence SEQ ID NO:37, or SEQ ID NO:38, and wherein the anti-CD123 scFv V L  domain comprises the amino acid sequence SEQ ID NO:42, or SEQ ID NO:43. 
 
     
     
       3. The polypeptide of  claim 1 , wherein the anti-CD123 scFv comprises the amino acid sequence SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51. 
     
     
       4. The polypeptide of  claim 1 , wherein the costimulatory signaling region comprises the cytoplasmic domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof. 
     
     
       5. The polypeptide of  claim 1 , wherein the CAR polypeptide is defined by the formula:
   SP-CD123-HG-TM-CSR-ISD; or 
   SP-CD123-HG-TM-ISD-CSR 
 wherein “SP” represents a signal peptide, 
 wherein “CD123” represents a CD123-binding region, 
 wherein “HG” represents and optional hinge domain, 
 wherein “TM” represents a transmembrane domain, 
 wherein “CSR” represents a co-stimulatory signaling region, 
 wherein “ISD” represents an intracellular signaling domain, and 
 wherein “-” represents a bivalent linker. 
 
     
     
       6. The polypeptide of  claim 1 , wherein the intracellular signaling domain comprises a CD3 zeta (CD3ζ) signaling domain. 
     
     
       7. The polypeptide of  claim 1 , wherein the polypeptide contains only an intracellular signaling domain or a co-stimulatory domain, but not both. 
     
     
       8. An isolated nucleic acid sequence encoding the recombinant polypeptide of  claim 1 . 
     
     
       9. A vector comprising the isolated nucleic acid sequence of  claim 8 . 
     
     
       10. A cell comprising the vector of  claim 9 . 
     
     
       11. The cell of  claim 10 , wherein the cell is selected from the group consisting of an αβT cell, γδT cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof. 
     
     
       12. The cell of  claim 11 , wherein the cell exhibits an anti-tumor immunity when the antigen binding domain of the CAR polypeptide binds to CD123. 
     
     
       13. The cell of  claim 11 , wherein the cell further comprise a second CAR polypeptide comprising a CD33 antigen binding domain, wherein the cell exhibits an anti-tumor immunity when both the antigen binding domain of the CAR binds to CD123 and the antigen binding domain of the second CAR binds to CD33. 
     
     
       14. A method of providing an anti-cancer immunity in a subject with a CD123-expressing cancer, the method comprising administering to the subject an effective amount of the cell of  claim 12 , thereby providing an anti-tumor immunity in the mammal. 
     
     
       15. The method of  claim 14 , wherein the immune effector cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell. 
     
     
       16. The method of  claim 14 , further comprising administering to the subject a checkpoint inhibitor. 
     
     
       17. The method of  claim 16 , wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof. 
     
     
       18. The method of  claim 14 , wherein the cancer comprises Acute Myeloid Leukemia (AML), blastic plasmocytoid dendritic cell neoplasm, hairy cell leukemia, and Acute Lymphoblastic Leukemia.